Optical scanners find use in performing detection for various experiments, assays and the like. They are often used in array analysis systems for detection of surface bound binding complexes in genomic and proteomic applications. In fluorescence optical scanners, fluorescence emitted from a sample that has been excited by a light signal is collected and detected.
Often, a user has little idea of the brightness of the fluorescence that will be emitted by a particular sample. Accordingly, the user does not know a priori how high or low to set the attenuation of an attenuator that controls the optical excitation signal power, that is, the signal power that reaches the sample. Further, the user does not know how high or low to set the gain of a detector that collects emitted fluorescence and produces a corresponding data signal.
Previously known optical system setup techniques include manually setting the sensitivity of the system where a user adjusts both the gain of the fluorescence detector and attenuation of the excitation light source. Typically, the user scans a sample in raster fashion to locate an element in the micro-array that is known to contain a concentration of a fluorophore that should produce a maximum fluorescence in response to the excitation signal. The user then re-scans the portion of the sample that contains this element and iteratively adjusts the sensitivity of the system until, in the judgment of the user, the corresponding data signal is sufficiently close to a maximum data signal value of the system. If the system has two channels, that is, produces excitation signals using two lasers of different wavelengths, the user re-scans the sample using the signal produced by the second laser and repeats the iterative, manual adjustment process to determine the appropriate sensitivity settings for the second channel. A user would further re-scan the sample for each additional channel.
The adjustment ranges of the attenuator or excitation source power and the detector are relatively large. Accordingly, manual adjustment of these components is time consuming, particularly since adjustment of either one of them may require a re-adjustment of the other. Thus, with manual scanning, the sample may be scanned many times to set the sensitivity of, or calibrate, the system. When multiple channels are used, more time is spent manually calibrating the system and the sample is scanned even more times, as discussed above.
If the excitation power and/or attenuator gain are set too low (i.e., detector sensitivity is too far reduced), the system may not accurately distinguish between different lower levels of emitted fluorescence. If the excitation signal power and/or detector gain are set too high, the system saturates. As stated in U.S. Pat. No. 6,078,390 to Bengtsson, assigned to General Scanning, Inc. (Watertown, Mass.), the general view is that such saturation results in a failure to make accurate measurements.
In response to such difficulties, the Bengtsson patent describes a scanning system and method of operation for automatically setting detection sensitivity. It employs an optical scanning system using a low-resolution scanning operation to automatically adjust the sensitivity of the system. The system performs a low-resolution scanning operation by scanning a line, automatically and iteratively setting the levels of excitation signal power and detector gain, skipping a plurality of lines and scanning a next line, adjusting the levels as appropriate, skipping a plurality of lines and scanning a next line, and so forth. After the system sensitivities have been set, the calibrated system then scans all the lines of the sample to collect data. The calibrated system thus scans for the first time the lines that were skipped during the low resolution “calibration” scanning operation.
For these skipped lines, photo-bleaching (i.e., weakening of fluorescent signal caused by exposure to excitation light) is avoided. With the other lines, however, the same problems encountered with manual scanning and tuning optical system attenuation or excitation system gain from photo-bleaching as a result of rescanning are encountered. The risk of damage to the sample is further increased when multiple channels are used.